Control of the density of surface-immobilized ligands or surface-binding sites is an important issue for the manipulation of surface properties and the development of sensors, array- or chip-based assays, and single-molecule detection methods. The density and chemical identity of surface-immobilized ligands can be used to control the wettability of surfaces, the selective adsorption of biological molecules, and the attachment and growth of cells on artificial surfaces. The signal level and response sensitivity that one observes from waveguide, interfacial fluorescence, or SPR-based sensors depends directly on controlling the density of reactive binding sites on the surface. For cooperative or multivalent binding of biological molecules to surface-tethered ligands, the density of ligands on the surface can significantly influence binding equilibria and energetics.
The control of ligand density on surfaces has been addressed in a number of ways. To produce gradients in ligand density on surfaces, one can generate a diffusion gradient of silane reagent in solution so that the target glass surface is exposed to a varying concentration of reagent, leading to a gradient in the density of bound silane ligands. This same approach can be implemented for generating gradients of organothiol ligands on metal surfaces. One clever method of producing a similar ligand density gradient on thin gold films involved the reductive desorption of thiol-bound ligands by applying an in-plane potential gradient across the film. Control of surface ligand densities and the preparation of density gradients can also be achieved by contact printing techniques by varying the concentration of ligand on the stamp or the contact time between the stamp and the surface. When characterizing the reactivity of isolated ligands on surfaces at the single-molecule level, it is important that the ligand spacing on the surface be controlled so that the response can be controlled. Widely spaced ligands correspond to very small (<10−6) fractions of a full monolayer and are challenging to generate in a controlled manner using the conventional or known techniques. Therefore, none of the existing techniques provides controllable and predictable placement of ligands at very low densities.